Table 2.

Real-time RT-PCR analysis of adrenal, anterior pituitary, and hypothalamic gene expression in PD2 and PD8 pups exposed to acute hypoxia

Tissue	Gene Symbol	Postnatal Day 2 Cycles to Threshold				Postnatal Day 8 Cycles to Threshold
		Baseline	4 h	P	2−ΔΔCt	Baseline	4 h	P	2−ΔΔCt
Adrenal	Star	23.71±0.23	23.65±0.10	0.800	0.81	23.98±0.15	22.84±0.14*	0.001	2.10
	Ldlr	31.40±0.40	29.72±0.41*	0.002	2.46	30.59±0.10	28.07±0.08*	0.001	5.46
	Cyp11a	23.20±0.10	23.44±0.08	0.097	0.76	23.55±0.08	23.88±0.27	0.282	0.90
	Cyp21a1	25.41±0.08	25.35±0.14	0.764	1.22	19.78±0.14	19.59±0.18	0.433	1.26
	Mc2r	31.89±0.18	31.94±0.09	0.799	ND	32.28±0.39	32.46±0.49	0.780	1.34
	Hif1a	26.83±0.13	27.11±0.09	0.110	0.78	26.27±0.04	26.77±0.21	0.061	0.67
	Npy	19.62±0.16	19.10±0.20	0.087	ND	19.67±0.49	19.43±0.40	0.716	ND
Anterior	Pomc	25.38±0.07	25.61±0.19	0.159	ND	22.02±0.22	22.01±0.33	0.980	ND
Pituitary	Crhr1	34.29±0.26	35.00±0.13	0.255	ND	29.42±0.05	30.50±0.17*	0.001	ND
	Hsd11b1	32.65±0.19	32.80±0.24	0.822	ND	32.08±0.25	32.63±0.22	0.171	ND
Hypothalamus	Crh	34.37±0.19	34.26±0.24	0.731	ND	33.44±0.18	33.62±0.16	0.461	ND
	Fos	26.96±0.11	26.25±0.17*	0.005	ND	26.76±0.07	26.57±0.17	0.317	ND
	Crhr1	27.43±0.07	27.73±0.10*	0.032	ND	29.02±0.09	29.11±0.09	0.475	ND
	Nr3c1 (GR)	23.73±0.06	23.89±0.09	0.190	ND	23.41±0.04	23.72±0.09*	0.011	ND
	Nr3c2 (MR)	27.33±0.06	26.94±0.22	0.115	ND	29.40±0.05	29.51±0.09	0.301	ND
	Hsd11b2	29.27±0.14	29.10±0.08	0.282	ND	29.44±0.17	29.53±0.08	0.640	ND
	Npy	22.35±0.15	22.70±0.08	0.060	ND	22.70±0.10	22.76±0.11	0.686	ND
	Npy1r	24.14±0.06	24.40±0.08*	0.020	ND	25.95±0.08	26.13±0.07	0.138	ND
	Actb	13.78±0.08	13.81±0.08	0.797	ND	16.57±0.08	16.73±0.11	0.282	ND

Whole adrenal glands (4–12 adrenals/sample) and anterior pituitary glands (2–6 pituitaries/sample) were pooled. Whole hypothalami were dissected and treated as one sample; n = 3–6 per tissue type for each gene of interest (at each age). RNA concentrations were normalized to 10–20 ng/μl, and each sample was assayed in triplicate for PCR analysis. A decrease in the cycle threshold (C_t) value at 4 h, as compared to baseline, translates into an increase in mRNA expression for that particular gene. Comparisons between age groups were not performed.

^*Significant difference in C_t value. Where indicated, the 2^−ΔΔCt equation was used to calculate the fold change in target mRNA expression (vs. baseline). The 2^−ΔΔCt equation uses a single reference gene (18S rRNA in this case) for the normalization of relative changes in target mRNA expression. See Table 1 for description of gene symbol abbreviations. ND, not determined.